Medical researchers and scientists have a need to isolate the various cells within whole blood samples in order to conduct tests and experiments.
Human blood for example has red blood cells and white blood cells. Red blood cells deliver oxygen from the lungs to the body tissues. Red blood cells are known as RBCs or erythrocytes (from the Greek erythos “red” and kytos for “hollow”). The white blood cells are produced in bone marrow and help defend the body from infectious disease and foreign materials. White cells derive their name from the fact that after centrifugation of a blood sample, the white cells are found in the Buffy coat, a thin layer of nucleated cells between the sedimented red blood cells and the blood plasma, which is white in color (or sometimes green, if there are large amounts of neutrophils in the sample, which are high in green. This green color is due to neutrophils having MPO, myeloperoxidase).
There are many different types of white blood cells. One primary technique to classify them is to look for the presence of granules, which produces the categories “granulocytes” and “non-granulocytes”. Granulocytes are a category of white blood cells, characterized by the fact that all types have differently staining granules in their cytoplasm on light microscopy. These granules are related to lysosomes found in some regular cells and primarily act in the digestion of engulfed invaders. There are three types of granulocytes: neutrophils, basophils, and eosinophils (named according to their staining properties). Non-granulocytes are a category of white blood cells which are characterized by the absence of granules in their cytoplasm. There are two types of non-granulocytes: lymphocytes and monocytes.
These categories can be further broken down into the following cellular types:
Neutrophils deal with defense against bacterial infection and other inflammatory processes and are usually the first responders to bacterial infection; their activity and death in large numbers form pus. Eosinophils primarily deal with parasitic infections and allergic disorders and an increase in them may indicate such. Basophils are chiefly responsible for allergic and antigen response by releasing the chemicals like histamine, causing symptoms of allergies. Lymphocytes are much more common in the lymphatic system. The blood has three types of lymphocytes: B cells: B cells make antibodies that bind to pathogens to enable their destruction. (B cells not only make antibodies that bind to pathogens, but after an attack, some B cells will retain the ability to produce an antibody to serve as a ‘memory’ system.) T cells: CD4+ (helper) T cells co-ordinate the immune response (they are what become defective in an HIV infection) and are important for defense against intracellular bacteria. CD8+ (cytotoxic) T cells are able to kill virus-infected cells. γδ T cells possess an alternative T cell receptor as opposed to CD4+ and CD8+ αβ T cells and share characteristics of helper T cells, cytotoxic T cells and natural killer cells. Natural killer cells: Natural killer (NK) cells are able to kill cells of the body which are not displaying a signal not to kill them, as they have been infected by a virus or have become cancerous. Monocytes share the “vacuum cleaner” (phagocytosis) function of neutrophils, but are much longer lived as they have an additional role: they present pieces of pathogens to T cells so that the pathogens may be recognized and killed, or so that an immune response may be mounted. Monocytes are also known as macrophages after they migrate from the bloodstream and enter tissue.
Since these cells are critical in fighting disease much research and testing is being conducted on these cellular components of blood.
In order to test a patient's blood, a sample is generally taken from a vein. Typically the samples are taken in volumes of 50 cc or more from donors. The whole blood can be placed into a centrifuge and the various blood components can be spun and layered over solutions that separate the cells by different densities such that a skilled technician using great skill and care can isolate certain cell types and withdraw them from the mixture using a pipette so a specific cell type within the whole blood sample can be studied.
This ability to isolate the cell types within blood samples is crucial in medical treatment and research.
The primary goal is naturally to collect as pure a sample of a certain cell type as possible. Along with that goal is another desire or goal to collect a sample that is not chemically altered during the collection process. In both of these goals the present techniques available are either extremely labor intensive and difficult to perform or alternatively if made simpler by the use of nonclonal antibodies, the antibodies can potentially alter the collected cells within a sample or contaminate the otherwise pure cell making it useless for serious research.
It is an objective of the present invention to provide a purification technique that leaves cells undamaged that can be performed using very simple steps requiring far less technical skill.
The technique further employs commonly used materials currently employed by technicians such that the new procedures will be easily adapted but will greatly simplify the task.